Through Phase I studies, we have identified five lead small molecule compounds with drug-like properties that activate innate immune response pathways and inhibit the replication of RNA viruses such as hepatitis C virus (HCV), influenza virus (FLU), and West Nile virus (WNV). There is a tremendous commercial demand for new antiviral products with novel mechanisms of action that target a broad spectrum of viruses. Most previous and ongoing pharmaceutical development programs involve screening for inhibitors of essential virus enzymes, with comparatively little investment in drugs that modulate the host immune response to infection. In this proposal, we focus drug development efforts on the host side of the virus-host interaction in order to discover and develop new antiviral products with novel mechanisms of action that are more effective and less sensitive to virus escape through mutation. In Phase 1, high-throughput screening of a 20,000-compound maximally diverse library, as well as a targeted library that was comprised of small molecules predicted by in silico modeling to bind to the RIG-I receptor, allowed identification of drug-like activators of an IRF-3 responsive promoter. Compound hits were counter-screened for target specificity and cytotoxicity, and validated for IRF-3 activation/nuclear localization, dose-dependent activation of interferon-stimulated gene (ISG)-reporter expression, and the ability to induce endogenous ISG protein expression in native 293 cells. Antiviral, mechanistic, and medicinal chemistry characterization of the hits resulted in the identification of five fully-validated lead compounds suitable for optimization and further pharmaceutical development. Phase 1 lead compounds decrease viral protein expression, RNA release, and infectious virus production in cell culture models of HCV, WNV and FLU. Genomic profiling demonstrates that lead agonists can activate a discrete antiviral gene program composed of a limited number of relevant innate immune targets. Based upon this initial effort, three Specific Aims are proposed for Phase 2. In Specific Aim 1, we will use strategically designed compound derivatives and biological and physiochemical assays to enhance lead compound potency and antiviral activity and identify lead structural classes and optimized drug leads for further characterization in exploratory tolerability and pharmacokinetic studies. In Specific Aim 2, the mechanism of action and scope of antiviral activity of lead structural classes will be characterized. Finally in Specific Aim 3, we will define the repeat-dose tolerability and in vivo antiviral properties of a small number of optimized drug leads in rodent models in order to support the selection of one lead and one back-up preclinical candidate for further commercial development. Key program innovations include the development of RIG-I-targeted antiviral drugs with a broad spectrum of action and drug-like physiochemical properties. The significance of the program derives from the global burden of viral diseases that may be addressed by these new drug candidates.